Aliquots of each culture were adjusted to equal OD600 values and then centrifuged. The supernatants were mixed with 5× SDS-PAGE loading buffer and boiled for 5 min. Aliquots (20 μl) of each sample were electrophoresed on a 10% SDS-PAGE gel, and the separated proteins were then transferred onto a nitrocellulose membrane. The membrane was blocked with TTBS, i.e., Tris-buffered saline-Tween 20 (0.05% vol/vol), and nonfat dry milk (5% wt/vol) for 1 h at room temperature, followed by probing with a rabbit poly-anti-CPB antibody (1:1,000 dilution) overnight at 4°C. Finally, bound antibody was detected with a horseradish peroxidase-conjugated secondary anti-rabbit antibody (Sigma-Aldrich), followed by addition of SuperSignal West Pico chemiluminescent substrate (Fisher Scientific).
Supersignal west pico chemiluminescent substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, Japan, China, Denmark, France, Panama, Canada, Israel, Sweden, India, Austria, Belgium
SuperSignal West Pico Chemiluminescent Substrate is a laboratory reagent used for the detection of proteins in Western blot analysis. It generates a luminescent signal when exposed to the target protein, allowing for visualization and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (144 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (125 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (117 mentions)
Nitrocellulose membrane, by Bio-Rad (84 mentions)
Ripa buffer, by Thermo Fisher Scientific (77 mentions)
Protease inhibitor cocktail, by Merck Group (71 mentions)
Protease inhibitor cocktail, by Roche (70 mentions)
Pvdf membrane, by Bio-Rad (63 mentions)
β actin, by Merck Group (56 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (54 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (51 mentions)
Ripa buffer, by Merck Group (49 mentions)
Chemidoc mp imaging system, by Bio-Rad (43 mentions)
Polyvinylidene fluoride membrane, by Merck Group (43 mentions)
Image lab software, by Bio-Rad (42 mentions)
β actin, by Cell Signaling Technology (41 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (35 mentions)
Trans blot turbo transfer system, by Bio-Rad (35 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (35 mentions)
β actin, by Santa Cruz Biotechnology (33 mentions)
2 543 protocols using supersignal west pico chemiluminescent substrate
1
Western Blot Analysis of CPB Protein
2
Endothelial Cell Culture and Signaling
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Pooled human umbilical vascular endothelial cells (HUVECs) and endothelial cell growth medium 2 (EGM2) were purchased from PromoCell (Heidelberg, Germany). Antibodies to ICAM1 and GAPDH were obtained from New England Biolabs UK Ltd. (Hertfordshire, UK). Anti-SOCS3 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). SuperSignal™ West Pico Chemiluminescent Substrate was from Fisher Scientific (Loughborough, UK). Secondary antibodies, anti-rabbit-IgG horseradish peroxidase, anti-goat-IgG horseradish peroxidase and anti-mouse-IgG horseradish peroxidase conjugates, were from Sigma-Aldrich Company Ltd. (Dorset, England). Forskolin, rolipram and N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) were obtained from Calbiochem (Paisley, UK). I942 (N-(2,4-dimethylbenzenesulfonyl)-2-(naphthalen-2-yloxy)acetamide) was purchased from MolPort (Riga, Latvia). Recombinant human interleukin 6 (IL6) protein and recombinant human soluble IL6 receptor α (sIL6Rα) proteins were purchased from R and D Systems (Abingdon, UK).
3
SDS-PAGE Protein Analysis Protocol
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Samples were run on 15 well Mini-Protean TGX gels (4 to 15%) under reducing conditions (5× reducing sample buffer, consisting of 14.5% SDS [wt/vol], 0.3 M Tris-HCl [pH 6.8], 50% glycerol, 0.015% bromphenol blue [wt/vol], and 20 mM dithiothreitol [DTT]). After protein transfer, nitrocellulose membranes were blocked with 10% milk for at least 1 h, and proteins were detected using the appropriate primary and HRP-conjugated secondary antibodies and SuperSignal West Pico chemiluminescent substrate (Fisher Scientific GmbH, Schwerte, Germany). Signals were detected using a Fusion FX7 imager (Peqlab Biotechnologie GmbH, Erlangen, Germany) and analyzed using the Image Studio Lite software (v5.2) and subsequent transformation in Microsoft Excel.
4
Protein Separation and Western Blotting
5
Immunoblotting of HIF-1α and ERK1/2
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MCF7 cell protein lysates were electrophoresed through a reducing SDS/10% (w/v) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against HIF-1 R & D Systems phosphorylated ERK 1/2 (E-4), ERK2 (C-14), TOMM20 (F-10) and β-actin (C2), all purchased from Santa Cruz Biotechnology. Proteins were detected by horseradish peroxidase-linked secondary antibodies and revealed using the SuperSignal west pico chemiluminescent substrate (Fisher Scientific).
6
Western Blot Protein Analysis
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Cell pellets were lysed using RIPA buffer (Sigma Aldrich) containing protease and phosphatase inhibitors (Roche, West Sussex, UK) and extracted proteins quantified using the BCA method (Smith et al, 1985 (link)). Protein extracts (100 μg) were loaded onto 12% (v/v) SDS-PAGE, followed by wet transfer to nitrocellulose. Following incubation in blocking buffer (5% milk and 3% BSA in TBS containing 0.05% Tween-20) for 1 h membranes were incubated overnight at 4 °C with anti-HOXD10 (1 : 250) or AMOT-p80 (1 : 250) antibodies. Membranes were incubated in anti-goat IgG HRP (1 : 50 000) or anti-rabbit IgG HRP (1 : 3000) antibodies for 1 h at room temperature and developed with SuperSignal west pico chemiluminescent substrate (Fisher Scientific).
7
Cell Lysis and Protein Extraction
8
Hedgehog Signaling Pathway Protein Analysis
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Commercially available primary antibodies used were as follows: Sonic Hedgehog, Cell Signaling Technology #2207 (Danvers, MA, USA); Patched, Biorbyt #157169 (San Francisco, CA, USA); SMO, #ab72130 (Abcam, Cambridge, UK); SuFu, Cell Signaling #2520; Gli, Abcam #ab134906; Gli2, #sc-271786 (Santa Cruz Biotechnology, Dallas, TX, USA); Gli3, Biorbyt #157158; Survivin, Santa Cruz #sc-17779; BCL-2, BD Pharmingen #556354 (San Jose, CA, USA); β-actin, Sigma #A5316. HRP-labelled second antibodies were from Cell Signaling.
For Western blot analysis, cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS), supplemented with aprotinin, leupeptin, pepstatin (Sigma), COMPLETE, and PhoStop (Roche, IN, USA). Total lysates containing 30 μg of protein were separated on SDS-PAGE gels and subsequently transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). Membranes were then subjected to probing with antibodies. Western blot signals were detected by using SuperSignal West Pico Chemiluminescent substrate (Fisher Scientific, Waltham, MA, USA) and exposed on films.
For Western blot analysis, cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS), supplemented with aprotinin, leupeptin, pepstatin (Sigma), COMPLETE, and PhoStop (Roche, IN, USA). Total lysates containing 30 μg of protein were separated on SDS-PAGE gels and subsequently transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). Membranes were then subjected to probing with antibodies. Western blot signals were detected by using SuperSignal West Pico Chemiluminescent substrate (Fisher Scientific, Waltham, MA, USA) and exposed on films.
9
Serum Antibody Screening with PERI-AMA-BSA
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For serum antibody screening, black 96-well microtiter plates (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) were coated at 1 μg mL−1 with PERI-AMA-BSA for 1 h at 37 °C in carbonate buffer (0.05 M carbonate-bicarbonate, pH 9.6). Then, the plates were blocked for 1 h at 37 °C with 3% non-fat dry milk in tris-buffered saline with 0.05% Tween-20 (TBST). After incubation for 1 h at 37 °C, TBST was removed and serum was loaded at a dilution of 1:100 in TBST and serially diluted. After another incubation for 1 h at 37 °C, plates were washed three times with TBST. Plates were then loaded with a secondary horse radish peroxidase labeled goat-anti-mouse antibody (Sigma) at 1:5000 in TBST and incubated for 1 h at 37 °C. Plates were washed and loaded with SuperSignal West Pico Chemiluminescent substrate (Fisher), incubated for 3 min, and then luminescent counts were recorded on a Victor3 Multilabel Counter (PerkinElmer, Waltham, MA, USA).
10
Chromatin Binding Assay Protocol
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For chromatin binding experiments, 15 μl LSS extract was supplemented with demembranated sperm nuclei (2500 sperm/μl) and incubated for 10 min at 21°C to allow chromatin assembly. For the time-course chromatin binding assays, AgNCs were added at the indicated timepoints. The reaction was then stopped at different times by diluting with the chromatin isolation buffer (50 mM HEPES–KOH, pH 7.8, 100 mM KCl, 2.5 mM MgCl2, 0.125% Triton-X100). The extracts were overlaid on top of 30% sucrose cushions in 1.5 ml protein low-retention tubes (Thermo Fisher Scientific). Samples were spun at 7800 rpm in HB-6 rotor for 30 min at 4°C. Chromatin pellets were resuspended in 10 μl Laemmli buffer, boiled at 90°C for 60 s and fractionated on a 3–8% gradient Tris–acetate gels (Invitrogen) according to standard procedures, followed by transfer of resolved proteins onto PVDF membranes (EMD Millipore). Following a 1 h block with 5% non-fat dried milk in PBS, membranes were incubated overnight at 4°C with one of the following primary antibodies: ORC1, ORC2, CDC6, MCM3 (52 (link)), CDC6, RPA32 and histone H3. HRP-conjugated secondary antibodies (anti-rabbit IgG HRP, anti-mouse IgG HRP, Fisher Scientific) and chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, 34077) were used.
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